Role of Long Non-coding RNAs on Bladder Cancer

Bladder cancer (BC) is the most common malignant tumor in the urinary system, and its early diagnosis is conducive to improving clinical prognosis and prolonging overall survival time. However, few biomarkers with high sensitivity and specificity are used as diagnostic markers for BC. Multiple long non-coding RNAs (lncRNAs) are abnormally expressed in BC, and play key roles in tumorigenesis, progression and prognosis of BC. In this review, we summarize the expression, function, molecular mechanisms and the clinical significance of lncRNAs on bladder cancer. There are more than 100 dysregulated lncRNAs in BC, which are involved in the regulation of proliferation, cell cycle, apoptosis, migration, invasion, metabolism and drug resistance of BC. Meanwhile, the molecular mechanisms of lncRNAs in BC was explored, including lncRNAs interacting with DNA, RNA and proteins. Additionally, the abnormal expression of thirty-six lncRNAs is closely associated with multiple clinical characteristics of BC, including tumor size, metastasis, invasion, and drug sensitivity or resistance of BC. Furthermore, we summarize some potential diagnostic and prognostic biomarkers of lncRNA for BC. This review provides promising novel biomarkers in early diagnosis, prognosis and monitoring of BC based on lncRNAs.


INTRODUCTION
Bladder cancer (BC) is one of the most common malignant tumors in the urinary system, and its incidence is increasing. It is estimated that 81,400 new cases and 17,980 deaths of BC in the United States in 2020 (Siegel et al., 2020). Clinically, 75-80% of BC patients exhibit moderately differentiated non-muscle-invasive bladder cancer (NMIBC) at the initial diagnosis, and the GRAPHICAL ABSTRACT | LncRNAs interacted with proteins in BC. (A) UCA1 interacts with C/EBPα. (B) Lnc-LBCS directly binds to hnRNPK and EZH2, and inhibits SOX2 transcription. (C) XIST-TET1-p53 pathway in BC. (D) DGCR5 promotes P21 transcription via interacting with ARID1A. (E) LSINCT5 promotes tumor progression by interacting with NCYM, and inhibiting GSK3β activity and promoting Wnt/β-catenin signaling activation. (F) GAS5 inhibits EZH2 transcription by interacting with E2F4. (G) AWPPH promotes cell proliferation, autophagy, and migration through binding to SMAD4 via EZH2. P indicates promoter. (H) LNMAT1 regulates CCL2 expression through interaction with hnRNPL. Protein-RNA interactions are important aspects of many cellular functions, and lncRNAs are involved in modulating BC through different molecular mechanisms, including binding to one or more protein partners.
remaining cases are muscle-invasive bladder cancer (MIBC). The standard treatment of NMIBC includes transurethral resection of bladder tumor (TUR-Bt), but high recurrence rate of BC after TUR-Bt and a subsequent tumor progression. The 5-year survival rate of patients with NMIBC is approximately 90%, while the 5-year survival rate of MIBC patients is only 20-40%, due to rapid disease progression and some early symptoms.
Long non-coding RNA (lncRNA) is defined as non-proteincoding RNA molecule longer than 200 nucleotides (Iyer et al., 2015). LncRNA plays an important regulatory role in epigenetic modification, transcription and post-transcriptional processing. Recent findings highlight that lncRNA contributes to the regulation of multiple signaling pathways in BC. LncRNA urethral cancer associated 1 (UCA1) was cloned from two human bladder transitional cell carcinoma cell lines. UCA1 is highly expressed in BC, and promotes progression, glycolysis, glutamine metabolism, migration and invasion of BC through the UCA1-mTOR/STAT3/miR-143/HK2 and UCA1/miR-16/GLS2 signaling network (Li et al., , 2015. Increasing evidence suggests that lncRNA exert their roles during the biological processes of tumorigenesis, tumor proliferation, differentiation, apoptosis, invasion, migration and stemness Nicolas, 2017;Zhang and Tang, 2018).
In this review, we focused on the expression, function and related genes or signaling pathways of lncRNAs, as well as the molecular mechanisms of lncRNAs and their clinical significance for diagnosis and prognosis in BC, which can provide new strategies and novel biomarkers for the diagnosis and treatment of BC based on lncRNAs.

IMPORTANCE OF NOVEL BIOMARKERS FOR BC
At present, diagnostic tests for BC include cystoscopy, urine cytology, FDA-approved markers, and non-FDA-approved markers. Four urinary FDA-approved commercial urinary biomarker tests are as follows: (1) bladder tumor antigen stat (BTA stat) and BTA TRAK; (2) Nuclear Matrix Protein 22 (NMP22) BC Test and NMP22 BladderChek; (3) uCyt+ assay; and (4) UroVysion assay. Several new biomarkers at the genomic, transcriptomic, and epigenetic levels have been developed, including: (1) ADXBLADDER, a novel ELISA test for Mini-chromosome maintenance 5; (2) Cxbladder Monitor; (3) XPERT BC, measure mRNA levels of ABL1, CRH, IGF2, UPK 1B, and ANXA 10; (4) UroMark assay; and (5) AssureMDX, test of the DNA methylation of OTX1, ONECUT2, and TWIST1 and somatic statuses of FGFR3, TERT, and HRAS in urine (Dangle et al., 2009;Tabayoyong and Kamat, 2018). Due to their poor sensitivity and specificity, cystoscopy and urine cytology are still used as the gold standard for the diagnosis of BC. Cystoscopy is the primary method for the diagnosis and monitoring of BC, and is not only invasive, entailing complications such as urinary tract infection or hematuria, but is also expensive (Casadevall et al., 2017). The urine cytology test is used to detect exfoliated malignant cells under the microscope, and is a non-invasive examination. It has high sensitivity and specificity for detection of highgrade BC; however, its sensitivity is only 4-31% for low-grade malignant tumor. In addition, false positive rate is as high as 12% due to inflammation and urothelial heterogeneity (Burchardt et al., 2000).
Early diagnosis of BC is essential for improvement of the prognosis and overall survival of patients. Given the current limited diagnostic methods, it is important to develop novel specific biomarkers as non-invasive tools that will facilitate accurate diagnosis and highly personalized treatment.

THE DEFINITION, FEATURES, AND CLASSIFICATION OF lncRNAs
Of the 3 billion base pairs in the human genome, only 1.5% are protein-coding sequences, and the rest are non-protein-coding sequences. There are many types of ncRNA, including shortstranded ncRNA and lncRNA. A greater proportion of lncRNA encoded by the human genome has tissue or cell type specificity, and is even related to specific stages of biological development. The definition of lncRNA was first proposed by Okazaki Y in 2002, mainly due to their discovery of a large number of lncRNA transcripts in large-scale sequencing studies of mouse cDNA libraries (Okazaki et al., 2002). LncRNA is defined as RNA longer than 200 nucleotides that not translated into proteins.
Anatomy of the global features of lncRNA suggests that lncRNA has some typical features, as follows: (1) lncRNA is usually larger than 200 nucleotides, (2) most of them have the eukaryotic structure of mRNA, the structure of promoter and polyA tail after RNA splicing, (3) contains few and long exons, (4) low expression, (5) less evolutionally conserved.
LncRNA has a variety of classifications according to its different characteristics. Currently, lncRNAs are mainly classified relying on four characteristics, including the location within the genome, the effect on the DNA sequence, the mechanisms of function, and targeting mechanisms of lncRNAs. According to the position of lncRNA in the genome and protein-coding genes, it is usually classified into five categories: sense lncRNA, antisense lncRNA, bidirectional lncRNA, intragenic lncRNA, and intergenic lncRNA. Based on the effect of lncRNA on DNA sequence, it can be divided into cis-lncRNAs and trans-lncRNAs. According to function mechanisms of lncRNA, it is fall into transcriptional regulation, post-transcriptional regulation, and translational regulation. Depending on the targeting mechanism of lncRNAs, it can be divided into signal, decoy, guide and scaffold (Ma et al., 2013).

EXPRESSION AND ROLE OF lncRNAs IN BC
To date, 120,353 human annotated lncRNAs have been collected by the LNCipedia database, and more than 70,000 published human and mouse lncRNAs have been annotated by NON-CODE . In general, lncRNAs show more tissue-specific patterns and higher expression variability than protein-coding genes in cell lines and tissues. A large fraction of lncRNAs preferentially localize in the nucleus, some are located in the cytoplasm, and a few are found both in the cytoplasm and the nucleus (Ponting et al., 2009). Nuclear lncRNAs are involved in diverse functions, such as histone modifications, chromatin remodeling, modulation of gene expression in cis or trans. Cytoplasmic lncRNAs can perform cytoplasmic functions, including posttranscriptional modification of mRNAs, interaction with proteins and miRNAs sponging.

Upregulated lncRNAs in BC
There are more than 100 dysregulated lncRNAs engaged in regulation of various biological functions of BC, such as cell proliferation, apoptosis, and metastasis. As shown in Table 1, forty-three upregulated lncRNAs involved in proliferation, migration, invasion, and cell cycle of BC, such as UCA1, long non-coding RNA 19 (H19), taurine up-regulated gene 1 (TUG1), and Calmodulin Like 3 Antisense RNA 1 (CALML3-AS1). Cytoplasmic lncRNAs can serve as oncogenes. UCA1, which is overexpressed in bladder cancer, specifically induces Glutaminase 2 (GLS2) by sponging miR-16, and can also activate AKT by recruiting E1A Binding Protein P300 (EP300) and lead to bladder cancer cell growth. UCA1 has a further oncogenic function by enhancing mTOR/STAT3/HK2 signaling pathway, which promotes Warburg effect of bladder cancer . Additionally, UCA1 is modulated by upstream molecules, such as bone morphogenetic protein 9 (BMP9), which then promotes tumor progression of bladder cancer.
LncRNAs can also be oncogene by regulating signaling molecules in the nucleus. Linc-UBC1 is upregulated and preferentially localizes in nucleus, which aggravates BC progression through altering polycomb repressive complex 2 (PRC2) complex localization by interacting with enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2) and SUZ12 polycomb repressive complex 2 subunit (SUZ12) (He et al., 2016). Similar to lncRNA upregulated in bladder cancer 1 (linc-UBC1), associated with poor prognosis of hepatocellular carcinoma (AWPPH) and non-coding RNA activated by DNA damage (NORAD) play regulatory roles in nucleus of bladder cancer cells.
A few lncRNAs can act as both cytoplasmic lncRNA and nuclear lncRNA. H19 promoter region can bind to CCCTCbinding factor (CTCF) and insulin like growth factor 2 (IGF2), and thereby regulating bladder cancer development in nucleus of BC cells (Takai et al., 2001). It was also reported that H19 promotes epithelial-mesenchymal transition and metastasis of bladder cancer via H19/miR-29b/DNMT3B signaling pathway in cytoplasm (Lv et al., 2017).

FUNCTIONAL MECHANISMS OF lncRNAs ON BC
Gene expression is modulated by lncRNAs at various levels. The mechanistic characterization of lncRNAs on bladder cancer is not well documented. It is partly due to the fact that lncRNAs harbor more cell type specificity and tissue type specificity, and generally low expression level. In spite of lncRNAs are less conversed among different species, there are similarities in functional mechanisms in bladder cancer. In this section, we highlight uncover molecular mechanisms from the following three aspects: lncRNAs by interacting with DNA, RNA and proteins.

LncRNAs Affect BC by Interacting With DNA
LncRNAs may anchor to promoters or regulators of target DNA to regulate gene expression in BC (Figure 1). LncRNA DBCCR1-003 as a tumor suppressor, derived from the DBCCR1 locus. DBCCR1-003 modulates the expression of DBCCR1 via DNA methylation in bladder cancer tissues and T24 cell lines. DBCCR1-003 can bind to DNA methyltransferase 1 (DNMT1), but the expression of DNMT1 remains unchanged when DBCCR1-003 is overexpressed in T24 cells. The results reveal that lncRNA DBCCR1-003 can modulate the expression of DBCCR1 by binding to DNMT1 and inhibiting DNMT1mediated methylation of DBCCR1 in BC . Linc-UBC1 can physically bind to EZH2 and SUZ12, key component of PRC2 complex. Some PRC2 target genes, such as Cyclin D2 (CCND2), plasminogen activator inhibitor type 2 (SERPINB2), Bone morphogenetic protein 2 (BMP2), Kruppel-like factor 4 (KLF4) and Homeobox A5 (HOXA5), are downregulated after knockdown of linc-UBC1. Moreover, H3K27 trimethylation in the promoter regions of CCND2, SERPINB2, the expression levels of BMP2 and HOXA5 decreased with the low expression of linc-UBC1. These results indicate that linc-UBC1 may partly modulate PRC2 complex localization by regulating histone H3K27 trimethylation with EZH2 and SUZ12 (He et al., 2013).
LncRNA HOX transcript antisense intergenic RNA (HOTAIR) modulates gene expression by binding to enzymes, such as EZH2, to inhibit histone modifications. HOTAIR mostly depends on EZH2, which is significantly increased in BC. HOTAIR originates from the HOX locus, which regulates HOX gene expression, differentiation and tissue homeostasis (Heubach et al., 2015). Additionally, the expression of HOTAIR is regulated by EZH2 after reduction of EZH2 in 5637 cells (Mónica et al., 2015).
Human H19 promoter contains large amount of methylcytosine in the CTCF binding site of and is abnormal hypomethylated in human bladder cancer. The sixth CTCFbinding site may serve as a significant regulatory domain, thereby switching H19 or IGF2 expression in human bladder cancer (Takai et al., 2001). Up to now, only a small number of lncRNAs has been demonstrated to participate in the regulation of BC through interacting with DNA.

LncRNAs Affect BC Through Interacting With RNA
Accumulating data indicate that both coding and non-coding RNAs can regulate one another by competing miRNA binding, known as competing endogenous RNAs (ceRNAs) (Salmena et al., 2011). ceRNAs can decoy miRNAs and protect their target mRNAs from degradation (Kartha and Subramanian, 2014). According to the ceRNA hypothesis, lncRNAs may exert their biological effects in BC by sponging miRNAs, which would in turn modulate the effect of miRNAs on their targets (Figure 2). H19 regulates the expression level of DNMT3B protein and epithelial-mesenchymal transition in bladder cancer. H19 can directly combine with the seed region of miR-29b-3p to sequester H19 by dual luciferase reporter assay, FISH and RIP assay. The expression of DNMT3B is repressed by miR-29b-3p in T24 cells. Collectively, H19 regulates the epithelial-mesenchymal transition and metastasis of BC through the H19/miR-29b-3p/DNMT3B axis (Lv et al., 2017). There is significant negative correlation between metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) and miR-124 in bladder transitional cell carcinoma tissues and normal mucosa samples. Foxq1, putative target gene of miR-124, is positively correlated with the expression of MALAT1 in bladder transitional cell carcinoma. Upregulation of MALAT1 promotes proliferation, migration, and invasion of bladder transitional cell carcinoma through the MALAT1/miR-124/foxq1 signaling pathway (Jiao et al., 2018). The exons 2 and 3 of UCA1 harbor miR-145 binding site and can suppress the expression of miR-145. ZEB1/2 and FSCN1 are engaged in migration and invasion of bladder cancer, and are the direct target genes of miR-145. These data suggest that UCA1 enhances BC migration and invasion through the miR-145-ZEB1/2-FSCN1 network . TUG1 and ZEB2 act as oncogene in bladder cancer. TUG1 contains the binding sites of miR-142, and TUG1 inhibits miR-142 expression in T24 and BIU-87 cells. These results imply that TUG1 enhances the proliferation and inhibits apoptosis of BC cells via the TUG1/miR-142/ZEB2 axis . LncRNA-activated by transforming growth factor-β (lncRNA-ATB) can promote cell proliferation, migration and invasion by capturing miR-126 in T24 cells. KRAS is a direct target gene of miR-126 and is inversed regulated by miR-126. These results indicate that lncRNA-ATB promotes cell proliferation, invasion and migration by decoying miR-126 in bladder cancer (Zhai and Xu, 2018).
Unlike sponge miRNAs, HOTAIR can repress miR-205 transcription by recruiting PRC2 and lysine-specific demethylase 1 (LSD1), and then recruiting H3K4me3 and H3K27me3 in miR-205 promoter region in HCV29 cells. Cyclin J is a putative downstream target gene of miR-205 in bladder cancer. These findings suggest that HOTAIR participates in the regulation of proliferation, migration and invasion of bladder tumor cells through the HOTAIR/miR-205/cyclin J signaling pathway (Sun et al., 2015).

LncRNAs Affect BC by Interacting With Proteins
Protein-RNA interactions are important aspects of many cellular functions, and lncRNAs are involved in modulating BC through different molecular mechanisms, including binding to one or more protein partners (Figure 3). UCA1 is upregulated by C/EBPα through the combination of core promoter region of UCA1 with C/EBPα in BLZ-211, 5637 and T24 cells. C/EBPα enhances UCA1 transcription was detected by qRT-PCR and luciferase activity. These results indicate that UCA1 increases cell viability and inhibits cell apoptosis through the UCA1 core promoter interacting with C/EBPα .
LncRNA low expressed in Bladder Cancer Stem cells (Lnc-LBCS) is markedly downregulated in bladder cancer tissues, and inhibits the self-renewal, tumor initiation, and tumor sphere formation of BC. Lnc-LBCS can act as a scaffold to activate the formation of hnRNPK-EZH2 complex by directly binding to heterogeneous nuclear ribonucleoprotein K (hnRNPK) and EZH2. Lnc-LBCS medicates SOX2 expression by forming hnRNPK-EZH2-SOX2 complex triplexes. Collectively, lnc-LBCS directly binds to hnRNPK and EZH2 and acts as a scaffold to induce complex formation to inhibit SOX2 transcription by regulating H3K27me3 . LncRNA X-inactive specific transcript (XIST) facilitates cell growth and supresses cell apoptosis by reducing p53 expression in T24 and 5637 cells. XIST can bind to ten-eleven translocation 1 (TET1), and TET1 can enhance p53 expression by combing with the promoter region of p53. These data show that XIST regulates cell proliferation, migration and apoptosis via the XIST/TET1/p53 pathway in BC (Hu et al., 2019).
LncRNA DiGeorge syndrome critical region gene 5 (DGCR5) is significantly downregulated in bladder cancer. Overexpression of DGCR5 represses cell proliferation, cell cycle progression, migration, invasion, and EMT while promotes cell apoptosis of bladder cancer. DGCR5 is require for AT-rich interaction domain 1A (ARID1A)-induced P21 expression in T24 and SW780 cells. This study demonstrate that DGCR5 promotes P21 transcription by interacting with ARID1A in BC (Fang et al., 2019a).
Long stress-induced non-coding transcript 5 (LSINCT5) directly interacts with NCYM, and inhibits GSK3β activity, leading to Wnt/β-catenin signaling activation and promoting EMT in bladder cancer cell lines . High GAS5 expression is correlated with poor prognosis in BC, and GAS5 effectively inhibits EZH2 transcription by binding and recruiting E2F4 to EZH2 promoter region, to promote BC cell apoptosis (Wang et al., 2018c). LncRNA AWPPH is highly expressed in BC and induces cell proliferation, autophagy, and migration, and restrains cell apoptosis by suppressing SMAD4 and EZH2 (Zhu F. et al., 2018). Long non-coding RNA lymph node metastasis associated transcript 1 (LNMAT1) promotes bladder cancer cells to lymph node and lymphangiogenesis in vivo. Chemokine (C-C motif) ligand 2 (CCL2) is necessary for LNMAT1-mediated lymph node metastasis, and is regulated by the LNMAT1-CCL2 promoter through directly interacting with hnRNPL in UM-UC-3 cells. Taken together, LNMAT1 regulates CCL2 expression through physically interaction with hnRNPL in BC .

LncRNAs COULD BE PROMISING DIAGNOSTIC OR PROGNOSTIC BIOMARKER IN BC
In recent years, accumulating evidence has demonstrated that abnormal expression of lncRNAs has crucial clinical significance for the diagnosis of BC, and is closely related to multiple clinical characteristics of BC, including tumor size, metastasis, invasion, tumor node metastasis (TNM) stage, diagnosis and prognosis ( Table 3).
In view of low expression, cell-specificity and tissue-specificity, lncRNAs represent favorable features to act as diagnostic or prognostic indicators for BC. Considering the low expression of lncRNA, the upregulated lncRNAs are more conducive to   (Duan et al., 2016;Ding et al., 2021). Taken together, UCA1 has relatively high sensitivity, specificity and AUC, and it can be regarded as the most potential diagnostic biomarker for bladder cancer.
As described above, it should be considered that lncRNAs can serve as feasible diagnostic and prognostic biomarkers. However, new lncRNA biomarkers used for clinical diagnosis must previously be carried out extensive studies in cells and clinical specimen. A single lncRNA may not be a reliable indicator for accurate and timely detection of bladder cancer. In this regard, additional systematic studies on lncRNAs will facilitate the diagnostic and therapeutic performance for bladder cancer.
As the current literature on lncRNA and bladder cancer pays more attention to the correlation between lncRNA and bladder cancer, there is still no certain lncRNA for clinical diagnosis, prognosis and subsequent therapy of bladder cancer. It help to combination of traditional indicators with novel lncRNA biomarkers for diagnosis and prognosis of bladder cancer.

DISCUSSION
Over the past decade, accumulating findings have uncovered that lncRNAs are crucial for initiation and progression of bladder cancer. Up to now, standard bladder cancer biomarkers are still very rare, owing to lack of the high sensitivity and specificity, and high costs of use. Due to the high recurrence and poor prognosis of bladder cancer, even after effective transurethral resection and systemic treatment, it is require to develop novel biomarkers for the early diagnosis and prognosis of bladder cancer. In this review, we summarize the expression, function, molecular mechanisms of lncRNAs, and the clinical significance of lncRNAs in the diagnosis and prognosis of bladder cancer. The molecular mechanisms of lncRNAs in bladder cancer have been explored, including lncRNAs interacting with DNA, RNA and proteins.
Circulating lncRNAs can be enriched in urine supernatant and plasma of bladder cancer, which may have a preferable potential for new bladder cancer tests. It has been suggested that aberrant expression of thirty-six lncRNAs is closely related to multiple clinical characteristics of bladder cancer. In the light of low expression, less evolutionally conserved, the upregulated lncRNAs represent favorable features to act as diagnostic or prognostic indicators for BC. Therefore, we screened potential clinical biomarkers among thirty up-regulated lncRNAs. Dissection of fifteen up-regulated lncRNAs associated with tumor size of bladder cancer, UCA1 has relatively high sensitivity, specificity and AUC, and it can be considered as the most potential diagnostic biomarker for bladder cancer.
Importantly, novel lncRNA biomarkers for clinical diagnosis must previously be carried out large studies in cells and clinical specimen. In this regard, extensive systematic studies on lncRNAs will facilitate the diagnostic and therapeutic performance for bladder cancer. It is worth noting that there is currently no any lncRNA can be applied to the specific diagnosis, prognosis and treatment of bladder cancer. In addition to mRNAs, microRNAs, circular RNAs and exosomes also play important roles in the development of BC. Our present knowledge indicates that combination of mRNAs, microRNAs and lncRNAs would hopefully better for improvement of early diagnosis and prognosis in bladder cancer.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.

AUTHOR CONTRIBUTIONS
H-JL, XG, and Z-KL were responsible for the framework and the writing of manuscript, and analysis of references. WQ, C-XH, and LX were mainly dedicated to searching and sorting of references. XZ and DZ were focused on the reference analysis. H-LC was mainly responsible for the revision and polishing of the manuscript. All authors have made great contributions.